Ongoing Oxidative Stress Causes Subclinical ... - Semantic Scholar

Original Research published: 14 March 2016 doi: 10.3389/fimmu.2016.00092

Ongoing Oxidative stress causes subclinical neuronal Dysfunction in the recovery Phase of eae Helena Radbruch1* , Daniel Bremer2 , Robert Guenther2 , Zoltan Cseresnyes2 , Randall Lindquist2 , Anja E. Hauser2,3 and Raluca Niesner2* 1 Department of Neuropathology, Charité – Universitätsmedizin Berlin, Berlin, Germany, 2 German Rheumatism Research Center (DRFZ) a Leibniz Institute, Berlin, Germany, 3 Immundynamics, Charité – Universiätsmedizin Berlin, Berlin, Germany

Edited by: Saparna Pai, The Centenary Institute, Australia Reviewed by: Nathan Karin, Technion – Israel Institute of Technology, Israel Anne Kathrin Mausberg, Heinrich Heine University, Germany *Correspondence: Helena Radbruch [email protected]; Raluca Niesner [email protected] Specialty section: This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Immunology Received: 17 October 2015 Accepted: 25 February 2016 Published: 14 March 2016 Citation: Radbruch H, Bremer D, Guenther R, Cseresnyes Z, Lindquist R, Hauser AE and Niesner R (2016) Ongoing Oxidative Stress Causes Subclinical Neuronal Dysfunction in the Recovery Phase of EAE. Front. Immunol. 7:92. doi: 10.3389/fimmu.2016.00092

Most multiple sclerosis (MS) patients develop over time a secondary progressive disease course, characterized histologically by axonal loss and atrophy. In early phases of the disease, focal inflammatory demyelination leads to functional impairment, but the mechanism of chronic progression in MS is still under debate. Reactive oxygen species generated by invading and resident central nervous system (CNS) macrophages have been implicated in mediating demyelination and axonal damage, but demyelination and neurodegeneration proceed even in the absence of obvious immune cell infiltration, during clinical recovery in chronic MS. Here, we employ intravital NAD(P)H fluorescence lifetime imaging to detect functional NADPH oxidases (NOX1–4, DUOX1, 2) and, thus, to identify the cellular source of oxidative stress in the CNS of mice affected by experimental autoimmune encephalomyelitis (EAE) in the remission phase of the disease. This directly affects neuronal function in vivo, as monitored by cellular calcium levels using intravital FRET–FLIM, providing a possible mechanism of disease progression in MS. Keywords: NOX, EAE/MS, intravital imaging, FLIM–FRET, calcium

INTRODUCTION Multiple sclerosis (MS) is a chronic neuroinflammatory disease, with most patients exhibiting a relapsing and remitting course of disease. The neurological damage is a consequence of a mainly T cell-driven immune reaction against myelin in the central nervous system (CNS) (1). Macrophages/ microglia, B, and T cells create an acute inflammatory setting, resulting in demyelination and neuronal damage. Most of the patients who experience a second episode develop further relapses. Despite the intensive analysis of the acute immune attack, only little is known about the processes going on at the lesion site after the initial insult (2, 3). Why and where do new relapses appear? What factors determine the chronicity of a lesion and the course of disease? “Old” lesions appear morphologically inert and are characterized by single perivascular T cells, minimal axonal damage in histological stainings with anti-amyloid precursor protein (APP) antibodies and a dominant fibrotic glial scar (4, 5). In contrast to the progressive disease phase, the inflammatory phase is well modeled by murine experimental autoimmune encephalomyelitis (EAE). In this mouse model using an immunization with MOG35–55 peptide, acute clinical signs remit after a few days and mice enter into a chronic phase with or without a residuum of neurological deficits (1). Reactive oxygen species (ROS) generated by invading and resident CNS macrophages have been implicated in mediating demyelination and axonal damage (6–8). In this

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Oxidative Stress in Chronic Neuroinflammation

study, we address implications of the ceased immune attack for the CNS tissue, beyond the fact that the majority of peripheral immune cells disappeared. In MS patients, we previously detected an ongoing over-activation of NADPH oxidases (especially NOX2) in blood monocytes during remission (8). Using intravital NAD(P)H fluorescence lifetime imaging in mice affected by EAE, during the inflammatory phase (onset and peak of the disease), we detected a massively increased amount of functional NADPH oxidases (NOX1–4, DUOX1, 2) within the CNS as compared to healthy controls (8). Using the same method, we investigated whether functional NADPH oxidases are still present in the CNS after recovery of EAE and, thus, whether oxidative stress is still ongoing in absence of peripheral infiltration of the CNS. We simultaneously monitor calcium concentrations in neurons using intravital FRET–FLIM-based neuronal calcium imaging to evaluate the reaction of the neurons on the altered CNS environment over the course of EAE development and remission. Thereby, we investigate whether in mice with clinical recovery morphologically inert appearing lesions exhibit residual inflammation, as reflected by increased oxidative stress and sub-clinical neuronal dysfunction, in order to better understand mechanisms of chronicity and disease progression in MS and related diseases.

of total cell number. All these findings are in line with the low clinical scores of the mice (between 0 and 0.5; Table 1) and are consistent with previous observations of cellular compositions after EAE recovery (10). Our results encompass two independent EAE experiments with a total number of n = 3 mice analyzed in remission phase (Table 1) and n = 5 analyzed healthy mice. Using intravital microscopy in CX3CR1+/− EGFP mice (n = 3) after EAE recovery, we could show a reduced overlap of 3.6 ± 1.8% between EGFP and i.v. injected sulforhodamin 101 (SR101), which labels astrocytes both in health and in peak EAE (8). These results are similar to the overlap measured in healthy CX3CR1+/−EGFP mice labeled by i.v. injection with SR101 (2.7 ± 1.1% overlap, Figure 1C).

Intravital Imaging Reveals Morphologic Features of EAE Remission in the CNS

We performed intravital imaging experiments in the brain stem of CerTN L15 × LysM tdRFP mice (neurons express the Ca2+ indicator TN L15, while predominantly LysM+ phagocytes express tdRFP) and of CX3CR1+/− EGFP mice (microglia/macrophages express EGFP, while predominantly astrocytes are labeled by SR101). Infiltration of the CNS by LysM+ cells is transient, and varies with the stage of disease. In health, practically no LysM+ cells are present except for few perivascular LysM+ microglia. During peak of EAE, many LysM+ cells are present within CNS lesions, and they mostly disappear during the remission phase. We could only identify isolated regions where LysM+ cells were present inside or in the close proximity to blood vessels or meninges (Figure 2A). In contrast to the peripheral immune cells, the inflammatoryinduced gliosis of CNS-resident cells [microglia and astrocytes having phagocytic capacity (8)] persists after EAE recovery (Figure 2B). We evaluated shape and function of astrocytes and microglia to test our hypothesis that the function of these CNS cells, in chronic neuroinflammation, has persistently (pathologically) elevated phagocytic features, even in the absence of peripheral immune cells. First, we quantitatively analyzed the shape of microglia, based on the fact that resting microglia, typical for healthy CNS, are highly ramified, whereas activated microglia, especially those having a phagocytic function, lose their cellular processes and adopt an amoeboid shape. The amoeboid shapes are expected to be found especially in the diseased CNS (10). We used Fourier coefficients to quantify and reproduce the ramified shape of microglia and to quantify their shape changes in the remission phase as compared to health and peak of the disease. Briefly, single microglia cells were segmented from intravital microscopy data acquired in the brain stem of healthy and EAE mice (in peak and remission phase). Six two-dimensional projections from each three-dimensional object (cell) were generated, and their shape was approximated by overlapping circles as displayed in Figure 2C. Each layer of circles is mathematically characterized by a scalar parameter called Fourier coefficient. Thus, the first Fourier coefficient defines the position of a cell, the second defines its dimensions by approximating it with a perfect

RESULTS Characterization of the Remission Phase in the CNS of Mice Affected by EAE

The grade of inflammation in brain stem of mice with EAE after clinical recovery (remission) was characterized and compared to animals at the peak of disease and to healthy controls. Our aim was to first characterize peripheral and CNS resident cellular compartments during the remission phase by means of intravital imaging and to corroborate previous results concerning the lack of overt inflammation in this phase.

Characterization of Cellular Markers in the CNS, during EAE Remission

It is widely accepted that in MS, inactive CNS lesions with no signs of immune infiltration are detectable. In our EAE model, some mice show a complete clinical recovery of EAE signs. We characterized these mice by FACS analysis of whole murine CNS (brain and spinal cord) and demonstrated that both monocytes/ macrophages (CD45highCD11b+ cells) and T cells (CD45highCD3+ cells) disappear from the CNS during the remission phase of EAE. Only 7.9 ± 2.8% of the isolated CNS cells were CD45highCD11b+ cells (macrophages/monocytes), comparable with healthy controls with 6.2 ± 2.4% (Figures 1A,B), whereas their frequency during onset and peak of EAE was previously shown to be strongly increased, to ~50% of the infiltrates (8–10). The majority of cells after EAE recovery were cells with characteristics of microglia: 72.5 ± 3.6% were CD45lowCD11b+ of which 95.2 ± 6.7% expressed CX3CR1. The overlap of CX3CR1 and tdRFP (LysM) was in both compartments under 5% (3.5 ± 3.2% for CD45highCD11b+ cells and 3.5 ± 3.1% for CD45lowCD11b+ cells). CD45highCD3+ cells – typically present during the peak of EAE (11) – mainly disappeared after EAE recovery, constituting only 0.2 ± 0.1% Frontiers in Immunology | www.frontiersin.org

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B

% of cells

ns.

CD45lhiCD11b+ CD45lowCD11b+ CD45hiCD11b+

healthy

CD3+

CD45

MP

CX3CR1

A

tdRFP

remission

CD45

% of cells

CD11b

CX3CR1+ in MP

tdRFP

CX3CR1+tdRFP+ in MP CX3CR1+ in MG

CX3CR1

MG

CX3CR1+tdRFP+ in MG

CD3 C

CX3CR1 SR101

FIGURE 1 | Peripheral immune infiltration of the CNS has largely resolved in the remission phase of EAE. (A) FACS analysis of CNS cells after recovery of EAE shows a low immune infiltrate with few monocytes/macrophages (CD45highCD11b+; MP) and CD3+ cells. Most of the CD45 expressing cells are CD45lowCD11b+ (microglia; MG). CD45highCD11b+ frequencies are comparable to healthy untreated mice (n = 5; ±SD). We applied an unpaired t-test to statistically evaluate the results. In the CD45highCD11b+ fraction (MP), only few cells express CX3CR1 but most of the cells in the CD45lowCD11b+ fraction (MG). The overlap of CX3CR1 and tdRFP was comparable in both cell types around 3% (n = 3; ±SD, clinical information listed in Table 1) (B) Exemplarily gating strategy of the FACS analysis of whole CNS in mice after recovery of EAE. (C) Projection of 3D intravital fluorescence image in the brain stem of a CX3CR1+/− EGFP mouse in health and during the remission phase of EAE. The astrocytes (and blood vessels) are labeled by i.v. injected SR101 (red), while the microglia are expressing EGFP (green). Scale bar = 50 μm. The colocalization of the EGFP and SR101 signals, i.e., overlap of the microglial and astrocytic markers, respectively, amounts to 3.6 ± 1.8%.

sphere, and the next Fourier coefficients define the number and length of cellular processes. Each cellular process is approximated by a set of spheres of various diameters, with the center on the surface of the most distant, previous sphere (Figure 2C). The higher the ramification and the length of cellular processes, the larger are the relative values of the high-order Fourier coefficients with respect to the second Fourier coefficient. We found a high shape similarity of microglia during the remission phase (118 cells) and of those imaged at the lesion site, at the peak of the


disease (57 cells). In comparison to resting microglia in healthy controls (71 cells), the similarity was rather low (Figure 2D). The third, fourth, and fifth Fourier coefficients show a significant difference (using an ANOVA test) both in remission and in peak as compared to healthy controls. The results encompass two independent EAE experiments with n = 3 healthy controls, n = 2 mice at peak EAE, and n = 4 mice during the remission phase. The findings of our intravital experiments demonstrate that in remission, after clinical recovery, microglia retain an

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TABLE 1 | Mouse strains, EAE data of the mice and mean NOX activation area values within lesions or gliosis/astrogliosis areas with SD per animal (6–20 areas within the brain stem per animal). EAE ID

Mouse strain

1 1 1 2 2 3 3 3 3 3 4 4 4 4 5 5

CX3CR1.EGFP CerTN L15 × LysM tdRFP CerTN L15 × LysM tdRFP CerTN L15 × LysM tdRFP CerTN L15 × LysM tdRFP CerTN L15 × LysM tdRFP CerTN L15 × LysM tdRFP CX3CR1.EGFP CX3CR1.EGFP CX3CR1.EGFP CX3CR1.EGFP CX3CR1.EGFP CX3CR1.EGFP CerTN L15 × LysM tdRFP CerTN L15 × LysM tdRFP CerTN L15 × LysM tdRFP

Healthy controls

Mouse strain

1 2 3 4 5

CerTN L15 × LysM tdRFP C57BL/6 CerTN L15 × LysM tdRFP C57BL/6 CerTN L15 × LysM tdRFP

EAE score at analysis time point

Maximum EAE score

Mean NOX activation area (%)

SD

1.5 1.0 0.5 2.5 0.5 2.0 2.0 1.5 0.0 0.0 0.0 0.0 0.5 0.0 0.5 0.5

1.5 1.0 2.0 2.5 1.5 2.0 2.0 1.5 1.5 1.0 2.0 1.5 2.0 2.0 3.5 3.5

17.88 13.20 8.15 16.99 9.91 10.65 7.18 11.61 9.27 8.77 11.53 9.71 12.19 – – –

5.13 1.06 2.25 9.02 3.97 0.51 1.42 1.34 3.25 3.97 5.38 2.79 4.65 – – –

Mean NOX activation area (%)

SD

0.37 2.84 0.47 0.60 2.08

0.13 0.29 0.09 0.28 0.91

We included five independent EAE experiments and five healthy controls for the intravital NAD(P)H–FLIM experiments.

activated morphology, suggesting that their function remains predominantly phagocytic despite the fact that clinical symptoms disappeared. Consistent with the results of shape analysis of microglia, the astrocytic network appears intact in healthy controls (n = 2), whereas during peak EAE (n = 4) and the remission phase (n = 3), it appears disrupted (Figure 2E). Additionally, the fine astrocytic processes completely disappear and are replaced by thick perivascular processes, while the astrocytic cell bodies adopt ameboid shapes (Figure 2E). A quantification of these observations is rather difficult. Even if a good segmentation of the single astrocytes and their processes is given, currently there is no available mathematical approach or set of mathematical parameters to summarize the complexity of the profound changes of the astrocytic network. However, altogether the observations regarding morphological modifications suggest that the astrocytes are also shifted toward a phagocytic function.

showed in intravital imaging experiments of mice affected by EAE, a concentration of ~200 μM of ROS is detectable in the brain stem, in EAE, using local ROS labeling with Amplex Red. In contrast, in healthy animals, we could not detect any ROS generation. As ROS molecules are highly reactive and diffusive, their detection is limited and the analysis of their cellular source practically impossible. We circumvent this disadvantage by detecting the catalyzer of ROS production, i.e., NOX enzymes, using NAD(P)H–FLIM in vivo. We previously showed that high ROS concentration in the brain stem of EAE animals correlates with the over-activation of NOX enzymes as detected by intravital NAD(P)H–FLIM (12). The fluorescence lifetime of NADPH bound to NADPH oxidases is ~3650 ps (12), differing from generally active NADH- and NADPH-dependent enzymes [fluorescence lifetime of NAD(P)H ~2200 ps]. The over-activation of NADPH oxidases is a prerequisite of oxidative stress – known to be one of the main causes of neuronal dysfunction in chronic neuroinflammation (6, 13). In healthy mice, intravital NAD(P)H–FLIM of the brain stem reveals predominantly metabolic enzyme activity (8). At peak of EAE, the lesion site is associated with vast areas of activated NADPH oxidases, leading to increased oxidative stress (8). Surprisingly, even if overt inflammation and the clinical symptoms disappear, a local activation of NADPH oxidases does not decline to levels found in healthy mice. While the area of NOX enzymes activation in the brain stem of healthy mice amounts in average to 1.8 ± 1.3%, the same average value at peak of the disease significantly increases eightfold to 15.6 ± 5.1% and declines only slightly to 9.4 ± 1% during the remission phase, still over fivefold higher than in healthy mice (Figures 3A,C).

Subclinical Neuronal Dysfunction Correlates with Oxidative Stress without Overt Immune Infiltration after Recovery of EAE

Altered morphology often indicates functional changes, but morphology is not a direct measure of the cellular function. To evaluate alterations in cellular function over the course of EAE, we used intravital NAD(P)H fluorescence lifetime imaging (FLIM), as previously described (8), to detect the over-activation of NADPH oxidases (NOX1–4, DUOX1, 2). As we previously


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healthy

A

remission EAE

peak EAE

Thy 1 LysM

B

CX3CR1

3.

5.

x x x cell process

2.

4.

cell body Fourier coefficients

D

rel. value of Fourier coefficients

C

1

* **

0,1

healthy

E

peak EAE healthy remission EAE

*

1

peak EAE

2

3

4

5

Fourier coefficient ID

6

7

remission EAE

SR101 FIGURE 2 | Intravital imaging reveals that remission in EAE correlates with lack of overt immune infiltration, with persisting disruptions of the microglial and astrocytic networks. (A) 3D intravital fluorescence images in the brain stem of CerTN L15 × LysM tdRFP mice in health (n = 5), at peak EAE (n = 6) and in the remission phase (n = 2). λexc = 850 + 1110 nm, λem = 525 ± 25 nm (Thy1-Citrine in neurons depicted in green), λem = 593 ± 20 nm (LysM tdRFP in phagocytes depicted in red), scale bar = 50 μm. (B) 3D intravital fluorescence images in the brain stem of CX3CR1+/− EGFP mice in health (n = 3), at peak EAE (n = 4) and in the remission phase (n = 5). λexc = 935 nm, λem = 525 ± 25 nm (CX3CR1+/− EGFP in microglia/macrophages depicted in green), scale bar = 50 μm. (C) Using higher-order Fourier coefficients, we describe the complex shape of microglia. The first Fourier coefficient describes the position of the cells, the second coefficient the sphericity of the cell body and starting from the third Fourier coefficient, the ramification of all cell processes is reproduced: the higher the values of high-order Fourier coefficients with respect to the second Fourier coefficient, the higher the degree of ramification and length of cellular processes of microglia. (D) The different shapes of the microglia, shown in (B), were classified in health (71 cells) at peak EAE (57 cells) and in its remission phase (63 cells). The difference between the values of the third, fourth, and fifth Fourier coefficients is significant between healthy controls and remission, but not significant between peak of EAE and remission of EAE. Statistical significance was determined by ANOVA (*p